| Scirpus yagara,as a traditional Chinese medicine,has the effect of breaking blood,activating Qi and relieving pain.Pharmacological studies have shown that the have anti-inflammatory,anti-tumor metastasis and cardiovascular and cerebrovascular protective effects.In recent years,domestic and foreign scholars have isolated a large number of chemical components from the tubers of Scirpus yagara,including isocoumarins,stilbenes,flavonoids,anthraquinones,etc.In this paper,sparstolonin B(SsnB),dehydrophyllodulcin(DPL)and sciryagarol Ⅰ(J1),the anti-inflammatory active ingredients from the tubers of Scirpus yagara,were studied.With the help of collagenase injection model of cerebral hemorrhage in rats,the protective effect of SsnB on secondary injury after intracerebral hemorrhage in rats was explored,and the possible mechanism was preliminarily explored.The in vitro metastasis model of tumor cells was induced by activating TLRs to explore the effect of DPL and J1 on inflammation-induced tumor metastasis.The studies in this paper mainly include the following sections:Part 1:Total synthesis,purity determination and solubility study of Sparstolonin B(SsnB)SsnB was originally isolated from the tubers of Scirpus yagara.But due to its low content in plants,difficult to extract and separate,high in cost,and poor in solubility,it is difficult to meet the needs of drug research and medicinal.Therefore,the total synthesis method reported in the literature was used in this experiment,and its structure was determined by TLC and LC-MS of the reference substance(plant origin);Then the purity was calculated by peak area normalization method by high performance liquid chromatography.Its solubility was further investigated in order to better administer the drug to animalsThe results showed that:1)About 1.1g of SsnB was synthesized by taking 10 g of 2-methyl furoate as the raw material after 13 steps of reaction,including Friedel-Crafts alkylation,Diels-Alder reaction,reduction reaction,etc.2)Light yellow fluorescence spots of the same color were observed at the corresponding positions of the chromatogram of the tested sample and the chromatogram of the control sample at 365 nm.3)In LC-MS,the liquid phase peaks at the retention time t=3.986 min,and the mass spectrometric analysis shows that the molecular ion peak is 267.0[M-H]-,the relative molecular weight of the compound is 268,and the compound is identified as SsnB together with TLC.4)The purity of 98.49%was determined by high performance liquid chromatography,which can be used for animal drug administration;5)The solubility results showed that SsnB was soluble in 1 mol/L of sodium hydroxide,soluble in dimethylsulfoxide and slightly soluble in tetrahydrofuran.Because of poor solubility,it could be supplemented by dimethylsulfoxide for dissolutionPart 2:Protective effect of SsnB on secondary injury after intracerebral hemorrhage in ratsPrevious studies have found that SsnB has a good inhibitory effect on brain injury of mice after intracerebral hemorrhage in the autologous blood model of intracerebral hemorrhage Compared with the autologous blood model,the cerebral hemorrhage model of collagenase injection in rats is more similar to the pathological and physiological changes of human intracerebral hemorrhage,which is more suitable for studying the protective effect and therapeutic effect of drugs on neurological function defects after intracerebral hemorrhage.Therefore,the collagenase model in rats with intracerebral hemorrhage was further verified.To investigate the effect of SsnB on the modified neurological severity score(mNSS)and the moisture content of brain tissue in rats with intracerebral hemorrhage at the 1st,3rd,5th and 7th days after the hemorrhage.Changes of inflammatory cytokines IL-6 and TNF-a in intracerebral tissue homogenates of rats with intracerebral hemorrhage were detected by ELISA.The results showed that:1)SsnB 25 mg/kg group could significantly improve the neurological defects of rats on the 1st,3rd and 5th days after modeling,while 10 mg/kg group could significantly reduce the neurological defects of rats with intracerebral hemorrhage on the 3rd and 5th days.2)Both 10 and 25 mg/kg doses of SsnB can significantly reduce the water content in the ipsilateral brain tissue of rats with cerebral hemorrhage;3)The expression levels of TNF-a and IL-6 were significantly inhibited in the 25 mg/kg group at 1st,3rd and 5th days after modeling.TNF-a expression was significantly inhibited in the 10 mg/kg group at 1st,3rd and 5th days after modeling,while no significant difference was found in IL-6 expression.Conclusion:10 and 25 mg/kg dose of SsnB bleeding in rats after 1st,3rd,5th days can play an important protective effect,the early inflammation plays a significant inhibitory effect.Its mechanism may be protecting the microglial cells in the brain,reducing the damage of neurons,and reducing the water content of brain tissue and the expression of inflammatory cytokines.Part 3:Effects of dehydrophyllodulcin and sciryagarol Ⅰ on inflammation-induced tumor metastasisFirst,the effects of DPL and J1 on the mutation rates of four types of tumor cells,including mouse melanoma cell B16,human liver cancer cell HepG2,human breast cancer cell MCF-7 and human lung cancer cell A549,were detected by MTT assay,so as to obtain the maximum test concentration of non-toxic compounds on tumor cells.And then LPS(TLR4 ligand)and Pam3csk4(TLR2 ligand)were used as inducers to study the effects of DPL and J1 on migration,invasion and adhesion of the four types of cells.The results showed that:1)The maximum reagent concentration of DPL and J1 cells was 5μM and 20 μM respectively.2)Both DPL and J1 can inhibit the migration and invasion of LPS-induced four tumor cells in a dose-dependent manner,as well as the migration and invasion of Pam3csk4-induced B16,HepG2 and A549 cells.3)5μM DPL can obviously reduce the adhesive of LPS and Pam3csk4-induced HepG2 cells to fibronectin.2 pM DPL and different doses of J1 can also obviously decrease the adhesive of LPS-induced HepG2 cells to fibronectin,but not to type Ⅰ collagen. |
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